6022421Southern Research Specialized Biocontainment Screening CenterVEE_AV3526_012013110A Cell-Based Counter Screen testing Compounds that Inhibit VEEV TC-83, in strain KU V3526Southern Research's Specialized Biocontainment Screening Center (SRSBSC)Southern Research Institute (Birmingham, Alabama)NIH Molecular Libraries Probe Production Centers Network (MLPCN)Assay Provider: Dong Hoon Chung, University of LouisvilleAward: 1 R03 MH087448-01A1 Venezuelan equine encephalitis virus is one of the Alphavirus species in the family of Togaviridae characterized by a non-segmented, positive sense strand RNA virus. VEEV, endemic to North, Central and South America, is an example of an arbovirus passing from a mosquito vector through rodent hosts that may result in epizootic subtypes causing disease in equines and humans. While there are various subtypes, human infection follows a similar route of acute symptoms of fever, headache, lymphopenia, myalgia and malaise but the severity may increase to encephalitis. Fatalities occur in approximately 1% of the patients that present with disease. Although recorded natural epidemics are rare, the Centers for Disease Control and Prevention and the National Institute of Allergy and Infectious Diseases have classified it as a category B priority biodefense agent in part due to its history as a biological weapon and the ease with which it may be cultured, manipulated and aerosolized. Treatment of VEEV is limited to supportive care since there are no drugs available, although two non-FDA-approved vaccines are available for at risk populations. The live attenuated vaccine, TC-83, is produced in Central and South America for use in horses, while an inactivated form of VEE, C-84, is licensed for use in horses in the United States. Neither the TC-83 nor the C-84 vaccine is FDA approved for human use in the United States, although military and laboratory personnel may receive vaccination through the USAMRIID Special Immunization Program. Continued work to construct an efficacious VEE vaccine with fewer adverse events than what is commonly reported for TC-83, led investigators at USAMRIID to construct a genetically engineered live-attentuated cDNA VEE vaccine, V3526. This vaccine is currently in phase II trials. However, the continued severity of the side effects and the inability to provide complete disease protection by the vaccines to-date necessitates additional work to develop efficacious prophylactics. Cell Culture: Vero 76 (CRL-1587, ATCC) were purchased from ATCC and maintained in 37C incubator with 5% CO2. The cells were cultured in a complete media (Minimum Essential Media with Earle's salt and 10% fetal bovine serum). Cells were passaged once a week and harvested from flasks using 0.05% trypsin-EDTA. Assay Media - Preparation of Complete DMEM media: 5mL Pen/Strep (Gibco, Cat. No. 15149) and 50mL of heat-inactivated FBS (Gibco, Cat. No. 10082147 ) was added to 500mL of Dulbecco's Modified Eagle Medium (Gibco, Cat. No. 11995-073).Virus : V3526 strain. Virus was rescued from BHK C-21 cells that were transfected with infectious V3526 RNA. The recued virus was amplified in BHK C-21 cells once and then used as a stock virus.Dose Response Compound Preparation: For dose response screening, compounds or carrier control (DMSO) were diluted to 3x in Complete DMEM media. Test compounds were serially diluted 1:2 resulting in an 8 point dose response dilution series. (final plate well concentration ranging from 50uM to 0.39uM and a final DMSO concentration of 0.25%). Thirty ul of each dilution was dispensed to assay plates (0.75% DMSO) in duplicate.Control Drug: The positive control drug for this assay, mycophenolic acid was solubilized in DMSO. It was diluted and added to the assay plates as described for test compounds. Final concentration for ribavirin was 10uM. All wells contained 0.25% DMSO.Assay Set up: Vero 76 cells were plated in 96-well plates at a density of 15,000 cells per well in a volume of 45uL of DMEM complete media. The cells were grown for 24 hours prior to testing in a 5% CO2, 37C cell culture incubator. V3526 VEEV virus, was diluted in cell culture medium to be 750 pfu/ 15uL ( 0.05 MOI) and then added to the plates at a volume of 15uL per well. The plates were incubated for 48 hours in a 37C incubator with 5% CO2 .Endpoint Read: Following the two day incubation period, the assay plates were equilibrated to room temperature for 10 min and an equal volume (90uL) of Cell Titer-Glo reagent (Promega Inc.) was added to each well using a Microflow (Biotek,VT) and plates were incubated for an additional 10 min at room temperature. At the end of the incubation, luminescence was measured using a Synergy4 Multimode plate reader (Biotek,VT) with an integration time of 0.2 s. Data Analysis: Results are reported as percent (%) CPE inhibition and were calculated using the following formula: % CPE inhibition = 100*(Test Cmpd - Med Virus)/(Med Cells - Med Virus). Four ribavirin positive control wells were included on each plate for quality control purposes. To quantify the viral cytopathic effect, IC50s were calculated for each substance using the 4 parameter Levenburg-Marquardt algorithm with the minimum and maximum parameters locked at 0 and 100, respectively.Possible artifacts in this assay include, but are not limited to, compounds that interfere with the luciferase reaction, absorb luminescence, or precipitate.Compounds showing at least 30% inhibition at any tested dose were considered active. Compounds that did not repeat activity were labeled inactive.The following tiered system has been implemented at Southern Research Institute for use with the PubChem Score. Compounds in the primary screen are scored on a scale of 0-40 based on inhibitory activity where a score of 40 corresponds to 100% inhibition. In the confirmatory dose response screen, active compounds were scored on a scale of 41-80 based on the IC50 result while compounds that did not confirm as actives were given the score of 0.588727Primary and Confirmatory Screen588719Cytotoxicity Counter Screen588723Summary AID11036Venezuelan equine encephalitis virus (strain 3526)http://www.southernresearch.orgSouthern Research Institute1IC50152% Inhibition @ 50 uM Rep 111550513% Inhibition @ 25 uM Rep 111525514% Inhibition @ 12.5 uM Rep 111512.5515% Inhibition @ 6.25 uM Rep 11156.25516% Inhibition @ 3.13 uM Rep 11153.13000011444092517% Inhibition @ 1.56 uM Rep 11151.55999994277954518% Inhibition @ 0.78 uM Rep 11150.779999971389771519% Inhibition @ 50 uM Rep 2115505210% Inhibition @ 25 uM Rep 2115255211% Inhibition @ 12.5 uM Rep 211512.55212% Inhibition @ 6.25 uM Rep 21156.255213% Inhibition @ 3.13 uM Rep 21153.130000114440925214% Inhibition @ 1.56 uM Rep 21151.559999942779545215% Inhibition @ 0.78 uM Rep 21150.7799999713897715216Verification4254321CR Plot LabelsConcentrationResponse02CR Plot Labels 2ConcentrationResponse01 R03 MH087448-01A12Phenotypic ScreenYesMultiplexingNoBSLBSL2Screening Concentration Range Max50Screening Concentration Range Min0.78Assay FormatCell-basedAssay TypeViability/ToxicityAssay MethodEnd-pointAssay DetectionBio-luminescenceUsed for Hit Validation?NoUsed during SAR?Yes13134190211026.23741.550.36-0.570.68195.6101.611-0.1120.8131.4141.5151.816Verified2012211313419031210010.97478.2586.9680.7770.8861.51089.41188.91296.31384.51466.71516.616Verified201221131341905129914.71460.3564.663672.980.31163.81247.7134.9142151.416Verified201221131341906129817.07366.3467.3565.361570.380.11066.61151.91217.5131.7141.815-0.816Verified2012211313419071210012.38566.5661.5748.6821.21170.51267.71360.81429.8153.516Verified201221131465094110220.6322.4417.2518.262679.981.9930.41011.11120.11220.5139142.9152.516Verified201221131465095129913.453132.14103.1557.6626.3713.484.710100.61176.91229.91315.61410.3152.316Verified201221